To mount an immune response, T lymphocytes must home from blood into lymph nodes, recognize specific antigens by direct contact with dendritic cells, proliferate, differentiate, exit the lymph node, and migrate to other lymph nodes as memory cells or to tissues as effectors. These processes involve motility, cell recognition dynamics, and Ca2+ signaling - events that were hidden from view until fairly recently. Two-photon (2P) microscopy now permits real-time visualization of living cells deep within lymphoid organs, revealing an elegant cellular choreography under basal conditions and during an immune response. Ion channels in T lymphocytes regulate the triggering, intensity and duration of Ca2+ signaling leading to downstream changes in gene expression and cell proliferation. In particular, a Ca2+ channel (Orai1) and a K+ channel (Kv1.3) are being developed as immunosuppressive targets for treatment of autoimmune disorders. The overall goals of this project are to investigate how the 'default' antigen search strategy of naive T cells is optimized, how tolerogenic B cells and regulatory T cells (Tregs) interfere with naive T cell activation, and how ion channels in T cells can be targeted for in vivo immunosuppression. Studies will be based on 2P imaging of human and murine immune cells in experimental models of the immune response. In Aim 1, new techniques for long-term cell tracking in intact tissues will be developed, and applied in 3 further subaims: (i) To explore whether T cells are attracted locally and dynamically toward DCs; (ii) Characterization of cellular interactions in a model of B cell-delivered gene therapy by tolerance induction, and (iii) Regulatory T cell suppression of naive T cell activation. Aim 2 focuses on the in vivo action of Kv1.3 and Orai1, with three subaims: (i) Development of two novel preparations for human cell immunoimaging. (ii) Imaging of effector T cells that cause inflammation and tissue damage in autoimmune disorders during treatment with a specific blocker of Kv1.3 channels in both human and murine models. (iii) Exploration of the functional roles of the Orai1 channel in vivo by specific inhibition of Ca2+ channel activity. Collectively, the proposed experiments probe the molecular and cellular mechanisms that modulate immune responses, and which represent potential therapeutic targets for improved treatment of inflammatory and autoimmune diseases.